不同浓度氯化铵诱导张氏细胞的缺氧诱导因子-1α基因表达通路的研究

Effect of different concentrations of ammonium chloride on expression of hypoxia-inducible factor-1α in Chang liver cells

目的探索不同浓度氯化铵（NH4C1）处理对Changliver细胞缺氧诱导因子（HIF）-1α及血管内皮生长因子（VEGF）基因mRNA和蛋白表达的影响。方法培养人正常Changliver肝细胞，用不同浓度NH4Cl（1．25mmol／L、2．5mmol／L和5mmol／L）持续处理，以正常培养的Changliver细胞作为对照组，分别在传至5代、10代及15代时提取RNA及蛋白质，应用荧光定量PCR、免疫组织化学方法和蛋白免疫印迹技术检测HIF-1α和VEGF基因的表达。结果qPCR结果：与对照组相比，NH4Cl处理组细胞HIF-1α mRNA表达量明显增加（5代、10代和15代组内比较F值分别为：90976．659，1300．218，1896．800，P值均〈0．05），VEGFmRNA表达量明显增加（5代、10代和15代组内比较，值分别为：3554．143，2381．321，2591．954，P值均〈0．05）；免疫组织化学检测结果：与对照组相比，NH4Cl处理组细胞HIF-1α蛋白表达量明显增加（5代、10代和15代组内比较F值分别为：376．709，1615．358，1350．120，P〈值均0．05），VEGF蛋白表达量明显增加（5代、10代和15代组内比较，值分别为：904．789，5105．186，8644．498，P值均〈0．05）；蛋白免疫印迹检测结果：与对照组相比，NH4Cl处理组细胞HIF-1α蛋白表达量明显增加（5代、10代和15代组内比较F值分别为：228．499，6051．974，183．219，P值均〈0．05），VEGF蛋白表达量明显增加（5代、10代和15代组内比较F值分别为：5549．429，40187．665，120982．183，P值均〈0．05）。结论氨可使Changliver细胞HIF-1α及其下游基因VEGF在细胞中的表达增加，引起假性低氧现象。

Objective To investigate the effect of treatment with different concentrations of ammonium chloride （NH4C1） on the mRNA and protein expression of hypoxia-inducible factor-1α （HIF-1α） and vascular endothelial growth factor （VEGF） in Chang liver cells. Methods Normal Chang liver cells were cultured and treated continuously with different concentrations of NH4Cl （1.25, 2.5, and 5 mmol/L）. The Chang liver cells cultured normally were used as controls. RNA and protein were extracted at the 5th, 10th, and 15th generations. Quantitative real-time PCR, immunohistochemistry, and Western blot were used to measure the expression of HIF-1α and VEGF. Results Quantitative real-time PCR showed that compared with the control group, the NH4Cl treatment group showed significant increases in the mRNA expression of HIF-1α and VEGF across the 5th, 10th, and 15th generations （F = 90976.659/1300.218/1896.800 and 41825.754/2381.321/2591.954, all P 〈 0.05）. The results of immunohistochemistry showed that compared with the control group, the NH4Cl treatment group showed significant increases in the protein expression of HIF-1α and VEGF across the 5th, 10th, and 15th generations （F = 376.709/1615.358/1350.120 and 904.789/5105.186/8644.498, allP 〈 0.05）. As was shown by the results of Western blot, compared with the control group, the NH4Cl treatment group showed significant increases in the protein expression of HIF-1α and VEGF across the 5th, 10th, and 15th generations （F = 228.499/6051.974/183.219 and 5549.429/40187.665/120982.183, allP 〈 0.05）. Conelusion Ammonia can increase the expression of HIF-1α and its downstream gene VEGF in Chang liver cells and cause the phenomenon of pseudohypoxemia.